Cardiogenesis involves the expression of many genes which encode proteins required for muscle activity. Evidence has accumulated that different actin isoforms are expressed at specific stages of heart development. Therefore,k during the formation of the embryonic heart and the transition from foetus to adult the expression of one gene within an actin multigene family may be substituted by that of another one, in keeping with the requirement of muscle structure. The fundamental goal of this project is to determine why actin isoforms are switched during development of the embryonic heart and replace other multigene family isoforms. Can isoactins substitute for each other during formation of the paired heart tubes, fusion of heart premordia and during torsion and formation of cardiac vessels? One way to test the function of an actin isoform during the elaboration of the mammalian heart will be to target an inappropriate isoform to embryonic cardiac myoblasts at the correct developmental window in the intact embryo. Therefore, cis-acting elements and dominant control regions of the mouse smooth muscle and cardiac actin genes will be used to provide developmental and cell type specific expression in a transgenic vehicle. The most direct way of defining the function of a contractile protein isoform in vivo will be to specifically mutate the contractile protein genomic allele of interest while leaving the rest of the genome intact. This would facilitate a direct experimental test of the gene's function by intercrossing heterozygous individuals. By applying the technique of homologous recombination to mouse actins in cultured embryonic stem (ES) cells, it should be possible to detect and or select clones of cells which carry mutations caused by homologous recombination within these genes. Intercrossing selected F1 heterozygous animals will provide a direct functional test of the requirement of these loci during embryogenesis. can isoactins substitute for each other during cardiogenesis? It will be possible to determine if myocardial cells recognize the absence of a key actin isoform, and whether these same cells have the plasticity to reprogram and express a substitute actin. If a developmental block is achieved by homologous recombination, it will be important to rescue the mutation by directing the re-expression of the wild type actin or a substitute actin through transgenic technology. In this manner the function of various actin binding domains might also be defined under in vivo conditions and their role int he looping and rotation of the embryonic heart.